Mycobacteria are a complex bacterial genus characterized by a unique cell wall rich in complex lipids, carbohydrates and polyketides. The most clinically significant member of this genus is Mycobacterium tuberculosis (TB) a deadly obligate human pathogen with an immense global burden. Despite decades of research effort against this pathogen, TB still infects one third of the world population. In order to develop new and innovative treatment strategies against TB, a better understanding of the physiology and pathogenesis of TB is needed. The advent of genomics and proteomics techniques has significantly boosted how we study TB in recent years. However, there is a lack of suitable complementary, rapid techniques to study the lipid, polyketide and carbohydrate (organic) pools that are known to play important roles in the physiology and virulence of TB and other pathogenic mycobacteria. Towards these ends, we have been developing new rapid analysis techniques to study mycobacterial lipids, polyketides and carbohydrates. We initiated the development of a simple complementary technique to quickly view the lipid and polyketide profiles of the TB bacilli using 13C enriched crude lipid extracts analyzed with minimal manipulation by 2D 1H-13C HSQC NMR analysis. Within these complex 2D HSQC maps it was possible to find distinct signals that could be used to identify and quantify the presence of key lipid species. We then showed that these lipid profiles could be used for observing changes in the cell wall due to drug treatment, gene mutation and changes in the physiological environment, species characterization and virulence factor expression. Importantly, the response of this analysis technique is equimolar between lipid species unlike mass spectrometric based lipidomic methods. This allows for rapid comparative analysis of several different lipid species within the same sample. However, before this method can be truly used for comprehensive and quantitative lipidomics studies, work is required to further refine and validate the method. This method development based R21 application addresses the studies required to finalize this technique so that it can be used in systems biology studies of mycobacteria, complementary to other lipidomic, genomic, proteomic and glycomic methods, and by the TB research community as a whole. The aims of this proposal are (i) Development and validation of a new rapid NMR based TB lipid profiling method; (ii) to perform 2 pilot studies designed to evaluate the robustness of the method and the quality of the data produced when the method is applied to studies of high clinical relevance. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis. M. tuberculosis is rich in lipids and organic materials that are essential for its survival and disease causing abilities. In this proposal it is planned to develop new rapid methods to study all these material simultaneously so that a new understanding of their role in the disease process can be deduced. [unreadable] [unreadable] [unreadable] [unreadable]